Bone enhancing composite

ABSTRACT

A bone-enhancing composite material comprising synthetic apatite and at least one supplementary bioactive agent selected from a biocompatible polymer and an anti-resorption agent added ab initio, methods of preparing said composite and uses thereof are provided. The physical and biological properties of the composite are controlled by the addition of specific supplementary bioactive agents as well as optional therapeutic agents. The composite may be used as a powder, a paste or an implant.

FIELD OF THE INVENTION

The present invention relates to the field of tissue engineering andmore specifically to a bone-enhancing composite comprising syntheticapatite and at least one supplementary bioactive agent selected from abiocompatible polymer and an anti-resorptive agent introduced into thecomposite ab initio suitable for use as a bone graft implant, a methodof preparing the composite and uses thereof.

BACKGROUND OF THE INVENTION

Tissue engineering may be defined as the art of reconstructing mammaliantissues, both structurally and functionally (Hunziker, Osteoart. Cart.,10:432-465, 2002). In general, tissue engineering includes the deliveryof a polymeric or ceramic scaffold that serves as an architecturalsupport onto which cells may attach, proliferate, and synthesize newtissue to replace tissue losses due to disease, trauma or age.Innovations in orthopedic surgery include a vast array of biomaterialsthat provide mechanical stability, controlled release of therapeuticagents and a scaffold for cell anchorage.

Bone

Bone is a unique type of tissue made up of an inorganic mineral phaseand cellular and extracellular matrix phases. Bone is a vital organ thatundergoes modeling and remodeling wherein old bone is lost (resorption)and new bone is formed (formation/replacement). Although bone has aninherent capacity for repair and regeneration when damaged by disease ortrauma, the renewed bone is often fragile and not weight bearing. Bonerestoration or replacement is a viable consideration in indicationsincluding osteopenia, osteoporosis, bone tumors, spinal fusion,fractures and non-union fractures.

Bone formation may be enhanced either by recruiting osteoblasts, thebone forming cells, or by inhibiting recruitment or activity ofosteoclasts, the bone resorbing cells. Osteoblasts and osteoclasts worktogether in a coordinated fashion to form and remodel bone tissue.

Many materials have been suggested for bone repair, specificallymaterials that avoid the harvesting problems associated with autologousmatter and the health risks associated with allogenic material.Inorganic material such as calcium phosphate has been utilized as boneand dental fillers (reviewed in LeGeros, Clin Orthop 395:81-98, 2002).Apatite, a particulate calcium phosphate, is particularly appealing byvirtue of the fact that it is the naturally occurring mineral componentin bone and teeth. Bone apatite exhibits low crystallinity due to thepresence of magnesium and carbonate (CO₃) ions. Lack of crystallinity inapatites is associated with increased solubility in vivo.Hydroxyapatite, in contrast, exhibits high crystallinity and representsa small component of natural bone. Synthetic bone substitute materialscomprising calcium phosphate or hydroxyapatite have been disclosed foruse as bone grafts implants and cements.

U.S. Pat. No. 4,880,610 teaches a method for producing an injectablecalcium phosphate mineral bone-like material using highly concentratedphosphoric acid, a calcium source and a neutralizing source, to whichvarious additives may be incorporated, including sugars or proteins suchas collagen, fibrinogen or elastin.

U.S. Pat. Nos. 5,650,176; 5,676,976 and 5,683,461 teach the synthesis ofreactive amorphous calcium phosphates (ACP) and their use for promotingbone growth. U.S. Pat. No. 6,214,368 discloses an injectable bonesubstitute comprising the reactive ACP, an acidic second calciumphosphate material and liquid to form an injectable paste capable ofhardening in vivo.

U.S. Pat. No. 5,281,265 discloses surgical cement comprising acalcium-based cementing component and a setting component capable ofhardening in vivo. U.S. Pat. No. 6,375,935 discloses a flowable calciumphosphate composition that hardens in vivo.

U.S. Pat. No. 5,071,436 discloses a spongy bone substitute matrixconsisting of glycosaminoglycans bonded to collagen together withhydroxyapatite. U.S. Pat. No. 6,118,043 discloses a porous bonereplacement material consisting essentially of calcium minerals havingan FGF polypeptide contained within.

U.S. Pat. Nos. 6,027,742 and 6,331,312 disclose a bioceramic compositecapable of resorption in the body. The composition comprises resorbablepoorly crystalline apatite (PCA) as a cement formed from amorphouscalcium phosphate, a promoter and a biocompatible supplementary materialselected from bioresorbable polymers or non-resorbable material, whichimpart a desirable biological, chemical or mechanical property. Thecomposite is prepared by combining the PCA with the supplementarymaterial.

U.S. Pat. No. 6,417,247 provides a composition comprising a polymer orpolymer solution that forms a gel under controlled parameters and aceramic matrix, the composition being fluid under non-physiologicalconditions and non fluid under physiological conditions. Thecompositions are prepared by mixing the ceramic component into a polymersolution.

EP 1208850A1 discloses a bone repair paste comprising an osteogenicpromoter, a calcium component and a viscosity-increasing agent. Thepaste is prepared by admixing the three components to yield asustained-release paste.

U.S. Pat. No. 6,231,607 discloses a novel bone substitute comprisinghydroxy apatite and both α- and β-tricalcium phosphate (TCP), preparedby microwave irradiation and subsequent sintering. The intermediatepowder material, resulting from microwave radiation, exhibits strongsimilarity to natural bone according to X-ray diffraction (XRD) andFourier transform infrared (FTIR) spectroscopy analyses. Derivatives ora fluid composition comprising the dry powder were neither taught norsuggested in that disclosure.

Anti-Resorptive Agents

Anti-resorptive agents such as bisphosphonates have been widely used toprevent bone resorption and lower fracture risk in patients withosteoporosis and other diseases exhibiting osteolytic processes. Certainbone implants and cements comprising an anti-resorptive agent have beendisclosed for filling bone voids and bonding prosthetic devices to bone.Denissen et al (Bone Miner. 25:123-134, 1994) describe the use ofbisphosphonate-impregnated ceramic hydroxyapatite implants for themaintenance of bone mass following tooth extraction due to oral disease.

U.S. Pat. No. 5,733,564 teaches a method of treating endo-ostealmaterials by immersion in a bisphosphonate solution to enhancebiocompatibility of prostheses. WO 00/47214 discloses anti-resorptivebone cements, comprising one or more anti-resorptive agents, preferablya bisphosphonate, useful for filling bone voids, bonding prostheticdevices to bone and for reconstructive bone surgery.

Existing bone graft implants, including pastes and cements, are preparedby admixing preformed calcium-based materials with a supplementarycomponent such as a polymer or an anti-resorptive agent. In general, thecalcium-based materials in the art, including synthetic apatites, areprepared using harsh conditions, e.g. low pH (phosphoric acid) or veryhigh temperatures (>450° C.), thus precluding the generation of acomposite incorporating such agents during the preparation steps. Theart has not heretofore provided synthetic apatite composite materialwherein a supplementary bioactive agent is included ab initio.

There remains an unmet medical need for a material having superiorbiological and physical properties for enhancing bone formation inorthopedic, periodontal and craniofacial indications.

SUMMARY OF THE INVENTION

Although numerous compositions comprising calcium phosphate minerals areknown in the art, none has proven entirely satisfactory in meeting thecriteria required for successful tissue engineering. The inventors ofthe present invention have found, quite surprisingly, that a novelcomposite material is produced by co-precipitating a liquid mixture ofcalcium, phosphate and carbonate ions with at least one amino acidmolecule in monomeric or polymeric form and at least one supplementarybioactive agent selected from a biocompatible polymer and ananti-resorptive agent that is present in the mixture ab initio. Thecomposite provides a superior material for orthopedic, periodontal andcraniofacial applications where bone enhancement is desired. Boneenhancement may be accomplished either by recruiting osteoblasts, thebone forming cells or by inhibiting recruitment and activity ofosteoclasts, the bone resorbing cells. The bone-enhancing composite maybe used per se or as a carrier to deliver therapeutic agents to the siteof the bone defect or lesion.

In one aspect the present invention provides a bone enhancing compositematerial comprising synthetic apatite and at least one supplementarybioactive agent selected from a biocompatible polymer and ananti-resorptive agent. Synthetic apatite is an artificial syntheticcalcium phosphate material prepared by precipitating calcium andphosphate from a solution. In one embodiment the synthetic apatitecomprises ionic calcium, phosphate and carbonate and at least one aminoacid in monomeric or polymeric form. In another embodiment the syntheticapatite is poorly crystalline apatite (PCA), providing a bonereplacement material superior to other synthetic apatite materials suchas hydroxyapatite or β-tricalcium phosphate due to its similarity tonatural bone and enhanced resorption capacity.

According to another aspect of the present invention, the at least onesupplementary bioactive agent is introduced during the preparation stepof the synthetic apatite ab initio. Without wishing to be bound by anytheory, the presence of a biocompatible polymer and/or ananti-resorptive agent during the formation of the synthetic apatitegenerates a unique composite material wherein the supplementarybioactive agent is intercalated or dispersed or distributed within thepoorly crystalline structure.

It is now disclosed that the attributes and desirable properties of thebone enhancing composite can be controlled by the addition of at leastone supplementary bioactive agent selected from a biocompatible polymerand an anti-resorptive agent in the process of forming the syntheticapatite. The supplementary bioactive agent is selected from a groupconsisting of biocompatible polymers and anti-resorptive agents.

According to one aspect the at least on supplementary bioactive agent isbiocompatible polymer. The composite comprising synthetic apatite and atleast one biocompatible polymer has attributes that make it particularlyadvantageous for supporting and promoting bone growth and repair invivo. Among the advantageous properties of the composite of theinvention:

-   -   a) The composite has superior physical properties, controlled by        various biocompatible polymers used in the preparation,        including poor crystallinity that highly resembles that of        natural bone, enhanced resorption and convenient formulation for        use in vivo.    -   b) The composite has superior biological properties, controlled        by various biocompatible polymers used in the preparation,        including controlled release of therapeutic agents,        biocompatibility, and the ability to promote cell growth,        proliferation, differentiation and migration.

The at least one biocompatible polymer, which may be natural orsynthetic, is selected to impart advantageous attributes to thecomposite. Without wishing to be bound by theory, the biocompatiblepolymers impart cohesive properties to the composite that may beoptimized for each of the diverse applications.

According to one embodiment of the present invention the biocompatiblepolymers that impart the desired properties are natural polymers ratherthan synthetic polymers. Examples of natural biocompatible polymersinclude polysaccharides and oligosaccharides. According to oneembodiment of the present invention the natural biocompatible polymer isa polysaccharide, preferably a sulfated polysaccharide such as aglycosaminoglycan selected from the group consisting of chondroitin4-sulfate, chondroitin 6-sulfate, hyaluronic acid, dermatan sulfate,keratan sulfate, heparin, heparan sulfate, sucrose octasulfate,perlecan, syndecan, glypican and combinations thereof. Heparin is meantto include the multiple molecular weight derivatives of heparinincluding very low molecular weight heparin, low molecular weightheparin, heparan, and heparin mimetics. Hyaluronic acid is meant toinclude cross-linked and non-crosslinked hyaluronic acid materials.Additional natural biocompatible polymers include starch, collagen,gelatin, glycogen, chitin, cellulose, keratins or combinations thereof.

In one embodiment the bone enhancement composite comprises syntheticapatite and heparin or a derivative thereof.

According to another aspect the at least on supplementary bioactiveagent is an anti-resorptive agent. The composite comprising syntheticapatite and at least one anti-resorptive agent has attributes that makeit particularly advantageous for enhancing and preserving bone in vivoand for applications such as securing prosthetic devices to bone andfilling lesions due to osteolytic processes such as metastases. Amongthe advantageous properties of the composite of the invention comprisingthe anti-resorptive agent:

-   -   a) The composite has superior physical properties, controlled by        varying anti-resorptive agents used in the preparation,        including crystallinity that resembles that of natural bone,        retarded resorption, good mechanical stability, structural        integrity and convenient formulation for use in vivo.    -   b) The composite has superior biological properties, controlled        by varying anti-resorptive agents used in the preparation,        including biocompatibility, local bone enhancement and increased        bone mass and inhibition of osteolysis.

According to one embodiment of the invention the at least oneanti-resorptive agent is a bisphosphonate or a pharmaceuticallyacceptable salt or ester thereof.

According to one aspect the present invention provides a compositecomprising synthetic apatite, at least one supplementary bioactive agentselected from a biocompatible polymer and an anti-resorptive agent,further comprising at least one therapeutic agent selected from thegroup consisting of antibiotics, antiviral agents, chemotherapeuticagents, anti-rejection agents, analgesics and analgesic combinations,anti-inflammatory agents, hormones, growth factors and cytokines.According to another aspect the at least one therapeutic agent is agrowth factor. According to one aspect the at least one therapeuticagent is selected from the group consisting of fibroblast growth factors(FGF) and bone morphogenetic proteins BMP), active fragments andvariants thereof. Preferably a therapeutically effective amount of FGFis provided, said FGF having the capacity to induce bone growth and orangiogenesis.

These and other features result in a composite exhibiting advantageousproperties including biocompatibility, biodegradability,osteoconductivity and osteoinductivity, controlled release oftherapeutic agents and ease of administration.

The bone-enhancing composite comprising synthetic apatite and at leastone supplementary agent selected from a biocompatible polymer and ananti-resorption agent is further characterized in that the composite hascrystallinity similar to that of natural bone. In one aspect of thepresent invention the composite has an X-ray diffraction (XRD) patternsimilar to that of poorly crystalline apatite and natural bone. Inanother aspect of the present invention the composite exhibits a peak of2 theta (2θ) at about 26° and an undifferentiated peak of 2 theta (2θ)at about 31° to about 33°.

According to another aspect the composite comprises at least onebioactive polymer and at least one anti-resorptive agent.

According to one non-limiting embodiment the composite is prepared bymicrowave heating the calcium and phosphate ions with the supplementaryagent using a procedure disclosed in U.S. Pat. No. 6,231,607. The powderresulting from that process consists of synthetic apatite or poorlycrystalline apatite (PCA) calcium phosphate aggregates having a size ofapproximately 0.45 μm to about 6 μm in diameter, more preferably about 1μm to about 4 μm in diameter. The aggregates are comprised of crystalsof approximately 0.20 μm to about 0.50 μm in size.

In one aspect the bone enhancing composite exhibits a calcium tophosphate ratio (Ca/P) similar to that of natural bone. Accordingly, thesynthetic apatite may contain cation or anion substitutions. In anotheraspect magnesium ions (Mg⁺⁺) and/or zinc (Zn⁺⁺) are added to partlyreplace the calcium ions, preferably Mg⁺⁺.

The bone-enhancing composite may be administered as a powder for certainbone disease and injury applications. In certain indications a fluid,semi-fluid or solid composition is preferred.

According to embodiment of the present invention a pharmaceuticalcomposition comprising the bone-enhancing composite is provided. Thecomposition may be fluid or semi-fluid. According to one embodiment ofthe present invention the composition is paste-like. According toanother embodiment the composition is an injectable paste. According toyet another embodiment the viscosity of the injectable paste is in therange of about 10 to about 500 poises, more preferably in the range ofabout 30 to about 200 poises, depending on the application.

According to one embodiment of the present invention the pharmaceuticalcomposition is fluid at temperatures below physiological conditions andnon-fluid at physiological temperatures. Preferably the composition gelsor hardens at about 35° C. to about 42° C. This particular property ofthe composition may be achieved by the addition of certain polymers orother additives to the composite of the invention.

In one aspect the composition comprises an additive that promotes insitu hardening of said composition within about 10 minutes to about 120minutes following injection. A non-limiting example of such materialsincludes collagens such as a gel forming soluble collagen disclosed inWO 00/47130, and non-polymeric compounds such as a non-polymeric estersor mixed esters of one or more carboxylic acids disclosed in U.S. Pat.No. 6,413,536. Another non-limiting example includes the addition ofcalcium sulfate or calcium phosphate compounds. The composition maycomprise about 5% to about 50% calcium sulfate.

Alternatively, in the reconstruction of structural tissues likecartilage and bone, certain applications may require the in vitromolding of the composition into three dimensional configuration articlesof varying thickness and shape. Accordingly, provided is an implantcomprising the composite of the invention further comprising a hardener,said implant having a specific shape including a sphere, screw, cube,rod, tube or plate.

In certain embodiments of the present invention a pharmaceuticalcomposition is provided comprising a composite material wherein thecomposite material comprises synthetic apatite, at least onesupplementary bioactive agent selected from a biocompatible polymer andan anti-resorptive agent, optionally further comprising at least onetherapeutic agent, further comprising a pharmaceutically acceptablecarrier or diluent. The pharmaceutical composition may optionallyfurther comprise hardening agents, the hardening agents having theability to induce setting of the composition into a solid article.

According to one embodiment of the present invention a pharmaceuticalcomposition is provided comprising synthetic apatite and heparincomposite, at least one carrier having sufficient fluidity to enableinjection of the composition to the site of treatment. According toanother embodiment of the present invention a pharmaceutical compositionis provided comprising synthetic apatite and heparin composite, at leastone carrier having sufficient fluidity to enable injection of thecomposition to the site of treatment and a therapeutically effectiveamount of at least one therapeutic agent selected from the groupconsisting of growth factors and their variants. According to yetanother embodiment of the present invention the at least one therapeuticagent is selected from the group consisting of fibroblast growth factors(FGF) and bone morphogenetic proteins (BMP), active fragments andvariants thereof. Preferably the growth factor has the capacity toinduce bone growth and or angiogenesis.

The pharmaceutical composition of the present invention is useful fortreating orthopedic, periodontal and craniofacial indications whereinthere is need to fill a void in a bone, to secure a prosthetic device ora need to delivery therapeutic agents to the bone or tissue in contactwith the bone. Tissue closely associated with bone includes, ligaments,tendons cartilage and muscle. In accordance with the invention use ofthe composite of the invention for the manufacture of a bone-enhancingmedicament is provided. The medicament is useful for treating diseasedor injured bone in orthopedic, periodontal and craniofacial indicationswherein the medicament is provided alone or comprising therapeuticagents that accelerate the healing rate and enhance the quality of boneformation or treat a disease or traumatized bone associated tissue.

Further provided is a method of preparing the bone-enhancing composite.The method comprises the following steps:

-   -   a) preparing a liquid mixture comprising ionic calcium,        phosphate, at least one amino acid in either monomeric or        polymeric form, carbonate, at least one supplementary bioactive        agent selected from a biocompatible polymer and an        anti-resorptive agent, optionally further comprising a        therapeutic agent;    -   b) subjecting said mixture to microwave irradiation;    -   c) quenching said irradiated mixture;    -   d) filtering said irradiated mixture so as to separate between        the filtrate and a cake;    -   e) drying said cake;    -   f) grinding said dried cake into a powder;

According to one embodiment of the present invention the above processmay further comprise the following steps:

-   -   g) sterilizing said powder;    -   h) wetting said sterilized powder with a solution optionally        comprising at least one therapeutic agent;    -   i) preparing said wetted powder for administration.

According to one embodiment of the present invention step (g) is carriedout in a manner that substantially retains the X-ray diffraction patternof the powder, preferably by ionization techniques, more preferably byγ-irradiation. The present inventors have found that followingsterilization by γ-irradiation the bone enhancing composite retains itsmolecular crystal structure, as determined by X-ray diffractionanalysis. Preferably the composite retains an X-ray diffraction patternhaving a peak of 2 theta at about 25° to about 26° and anundifferentiated peak of 2 theta at about 31° to about 33°. Thebone-enhancing material may be sterilized by thermal sterilization,preferably at about 140° C. to about 160° C. for at least 30 minutes,and retain its XRD.

According to one embodiment of the present invention step (h) of theabove method is performed using a pharmaceutically acceptable liquidsuch as water or a physiological fluid. The liquid is added in asufficient amount to permit wetting and dispersion of the powder to forma wetted mixture having the consistency of a paste, cement or putty. Theliquid may advantageously comprise at least one therapeutic agentselected from antibiotics, antiviral agents, anti-rejection agents,analgesics and analgesic combinations, anti-inflammatory agents,hormones, growth factors, cytokines and chemotherapeutic agentsincluding anti-cancer compounds.

The therapeutic agents, for example, growth factors, angiogenic factors,and the like, are likely to encourage a more rapid growth of the cellswithin the implant, or a more rapid vascularization of the implant Suchfactors may be too small to be effectively retained within the matrixand hence are introduced in the form of slow-release orcontrolled-release formulations into the matrix to provide for theireffectiveness.

In yet another embodiment a method for treating orthopedic, periodontaland craniofacial indications comprising administering to a subject inneed thereof a therapeutically effective amount of a compositioncomprising synthetic apatite, at least one biocompatible polymer,optionally further comprising at least one therapeutic agent.

Further provided is the use of a composite comprising synthetic apatiteand at least one supplementary bioactive agent selected from abiocompatible polymer and an anti-resorptive agent, optionally furthercomprising a therapeutic agent for the preparation of a medicament forthe treatment of orthopedic, periodontal and craniofacial indications.

Depending on the indication the bone-enhancing composite may be used perse or in a wetted form. According to one embodiment the bone substituteis a powder and is used per se. According to one embodiment thepaste-like material is administered directly to a bone defect. Accordingto one currently more preferred embodiment the paste-like material isinserted into a syringe for local administration. According to oneembodiment the paste-like material hardens in situ in about 10 minutesto about 120 minutes following injection. Alternatively, the paste-likematerial hardens in vitro to form a molded implant.

Furthermore, the composite may be used as a coating on synthetic orother implants such as pins and plates, for example, in hip replacementprocedures. Thus, the present invention further provides implants ormedical devices coated with the matrix of the invention. In anon-limiting example the bone enhancing composite comprising syntheticapatite and at least one anti-resorptive agent may be used to reduce theincidence of osteolytic debris induced by the wear and corrosion oforthopedic prostheses.

Yet another currently preferred embodiment of the present inventionprovides a kit comprising the disclosed bone graft composition, wherethe dry and liquid components may be present in separate containers inthe kit, or some of the components may be combined within one container.

These and other aspects of the present invention will be apparent fromthe description, figures and claims that follow.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1G show the X-Ray diffraction patterns of the bone enhancingcomposites comprising synthetic apatite and a biocompatible polymer orsynthetic apatite and an anti-resorptive agent.

FIG. 2 shows the particles of the bone-enhancing composites as seen inSEM (scanning electron microscope). FIG. 2A shows bone-enhancingmaterial without additives. FIG. 2B shows synthetic apatite-heparin (0.5ug/ml) composite, FIG. 2C shows synthetic apatite-heparin (100 ug/ml)composite. All figures are 39570× magnification.

FIG. 3A shows the results of the adsorption assay and FIG. 3B shows theresults of the proliferation assay for FGF on synthetic apatite, per seor composite. Proliferation was tested on FGFR1 expressing FDCP cells.FIG. 3C shows release of FGF from synthetic apatite-heparin compositesafter 3 days. FIGS. 3D and 3E show sustained release of FGF fromsynthetic apatite-heparin composites.

FIGS. 4A and 4B show histological sections of new bone formation in arat tibia model using a synthetic apatite-SOS composite.

FIGS. 5A and 5B show histological sections of new bone formation in arat tibia model using a using a synthetic apatite-heparin composite.

FIGS. 6A through 6D show histological sections of new bone formation ina rat tibia model. FIG. 6A shows bone formation in an untreated hole,FIG. 6B shows new bone surrounding a commercially available calciumphosphate ceramic. FIG. 6C shows the new bone surrounding the syntheticapatite particles while FIG. 6D shows new bone surrounding syntheticapatite-heparin composite particles. FIG. 6D shows full integrationbetween new bone and the implanted particles.

FIGS. 7A and 7B show histological sections of new bone formation in arat tibia model using a using a synthetic apatite-heparin compositefurther comprising a calcium sulfate hardening agent.

FIGS. 8A and 8B show histological sections of calvarial bone treatedwith synthetic apatite alone (8A) or synthetic apatite-alendronatecomposite (8B) stained for osteoclasts using TRAP assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a biocompatible compositecomprising synthetic apatite and at least one supplementary bioactiveagent selected from a biocompatible polymer and an anti-resorptive agentuseful as a bone enhancing implant.

In principle, an ideal bone enhancing material will exhibit thefollowing properties:

-   -   Biocompatible: minimal toxicity and maximal resemblance to        natural bone.    -   Practical: convenient for use by the medical practitioner.    -   Resorbable: capacity to biodegrade in the host over time.    -   Osteoinductive: capacity to induce regeneration or enhancement        of functional bone.

In addition, the bone enhancing material may also exhibit the followingproperties:

-   -   Osteoconductive: provide a microenvironment beneficial to        attachment, migration and proliferation of cellular elements        involved in bone growth.

The present invention provides a bone-enhancing composite exhibiting theaforementioned advantageous properties.

Bone enhancement may be accomplished either by recruiting osteoblasts,the bone forming cells or by inhibiting recruitment or activity ofosteoclasts, the bone resorbing cells. Accordingly, a bone-enhancingmaterial may stimulate de novo bone formation or may inhibit boneresorption.

Definitions

For convenience and clarity certain terms employed in the specification,examples and claims are described herein.

The term “synthetic apatite” refers to an artificial synthetic calciumphosphate material prepared by precipitating calcium and phosphate froma solution. Synthetic apatite may further comprise other materialsincluding carbonate ions, and magnesium or zinc.

“Poorly crystalline apatite” and “PCA”, refer to synthetic poorlycrystalline apatitic calcium phosphate. PCA has substantially the sameX-ray diffraction spectrum as natural bone. The spectrum is generallycharacterized by two peaks in the region of 2 theta 2θ at 25°-35°, withone centered at about 25° to about 26° and the other centered at about32°.

The term “biocompatible” as used herein refers to materials havingaffinity with living tissues, low toxicity and no unacceptable foreignbody reactions in the living body. In one aspect “biocompatible polymer”refers to a biodegradable polymer.

The term “osteoconductive” as used herein refers to materials thatprovide a microenvironment that is advantageous to the healing ofdiseased or damaged bone. Preferably, the composite of the inventionprovides a milieu that is advantageous to the infiltration andproliferation of cells involved in the process of bone repair.

A “composite” as used herein refers to a material that is made up of twoor more distinct elements. The composite of the invention is unique inthat it comprises a mineral phase and an organic phase that areintercalated or interdispersed ab initio.

A composite or composition considered “resorbable” is soluble anddegrades in vivo. Preferably, the material remains intact throughout theinitial healing of the bone and degrades slowly as the host's cellsinvade and proliferate within the area of the implant. “Biodegradable”refers to the resorption of the composite or composition within the hostin a manner that is non-toxic and non-immunogenic to the host.

The term “fluid” as used herein is intended to describe a compositionhaving sufficient viscosity so as not to disperse from the space it isintended to fill, yet having a viscosity low enough to be able to beadministered via syringe.

The term “viscosity” refers to the property of resistance to flow in afluid or semi-fluid. Viscosity is measured in a unit known as a poise.Suitable viscosities of the final solution mixture of the pharmaceuticalcomposition for each particular application may readily be establishedby the skilled person, but will generally be in the range of about 10 toabout 500 poises, preferably about 30 to about 200 poises.

This term “implantation” refers to the insertion of the composition ofthe invention into a patient, whereby the implant serves to replace,fully or partially, tissue that has been damaged or removed. Anotheraspect of implantation is also taken to mean the use of the composite asa vehicle to transport therapeutic agents to a certain site in apatient. In this aspect there is also included the adsorption onto thecomposite or of a therapeutic agent selected from growth factors,cytokines, chemotherapeutic drugs, enzymes, anti-microbials,anti-inflammatory agents. A fluid, semi-fluid or solid material may beimplanted.

The term “injection” refers to the insertion of a composition of theinvention into a mammal using a syringe or other device which allowsadministration of the composition directly to the site of treatment.Another aspect of injection is also taken to mean the use of thecomposite as a vehicle to transport therapeutic drugs and therapeuticagents to a certain site in a patient. In this aspect there is alsoincluded the introduction into the composite of a therapeutic agentselected from growth factors, cytokines, enzymes, anti-microbials,anti-inflammatory agents and chemotherapeutic agents such as anti-cancerdrugs.

Therapeutic agents including growth factors, angiogenic factors, and thelike, are advantageous to encourage a more rapid growth of the cellswithin the composite, or a more rapid vascularization of the materialthus reducing the healing time. Such factors may be too small to beeffectively retained within the composition and hence may be introducedin the form of slow-release or controlled-release formulations into thecomposite to provide for their effectiveness.

Chemotherapeutic agents include a variety of chemical compounds thatprevent or treat tumors and their metastases.

The inventors of the present invention have found that a novel compositematerial is produced by co-precipitating a liquid mixture of calcium,phosphate and carbonate ions with at least one amino acid molecule inmonomeric or polymeric form and at least one supplementary bioactiveagent selected from a biocompatible polymer and an anti-resorptive agentthat is present in the mixture ab initio.

Biocompatible Polymers

According to one embodiment of the present invention the compositecomprises at least one biocompatible polymeric agent that modulates thephysical and biological properties including crystallinity, surfaceadhesion, cohesion, ability to maintain cell growth and proliferation,and binding and retention of therapeutic agents for controlled release.

These biocompatible polymers include materials belonging to the familyof polysaccharides, anionic polysaccharides, glycosaminoglycans, orsynthetic biocompatible polymers, including hyaluronic acid, pectin,alginate, galactans, galactomannans, glucomannans, polyuronic acids,heparin, dextran sulfate, dermatan sulfate, heparin, heparan sulfate,keratan sulfate, hexuronyl hexosaminoglycan sulfate, chondroitin4-sulfate, chondroitin 6-sulfate, dermatan sulfate, keratan sulfate,perlecan, syndecan, glypican and PEG and combinations thereof

According to one embodiment, the composite is prepared using abiocompatible polymer wherein the biocompatible polymer is heparin or aheparin derivative or heparin mimetic. “Heparin mimetic” as used hereinincludes polysulfated sugars, such as polysulfated monosaccharides,polysulfated disaccharides and polysulfated oligosaccharides, includingsucrose octasulfate, inositol hexasulfate, and many other polysulfatedsugars that may act as analogs of low molecular weight heparins may beused to substitute for sulfated polysaccharides. For example, U.S. Pat.No. 6,143,730 discloses sulfated oligosaccharides comprising from 3 to 8monosaccharide units.

An “anionic polysaccharide” as used herein, is a polysaccharide,including non-modified as well as chemical derivatives thereof, thatcontains at least one negatively charged group (e.g., sulfate groupswhich are negative at neutral pH, and carboxyl groups at pH values aboveabout 4.0) and includes salts thereof, such as sodium or potassiumsalts, alkaline earth metal salts such as calcium or magnesium salts.Non-limiting examples of anionic polysaccharides include pectin,alginate, galactans, galactomannans, glucomannans and polyuronic acids.

Anti-resorptive Agents

According to one embodiment the present invention is directed to acomposite comprising synthetic apatite and at least one supplementarybioactive agent wherein the supplementary bioactive agent is ananti-resorptive agent. The composite is useful as an implant fortreating bone disorders and diseases or as cement to anchor prosthesesin certain orthopedic and dental indications.

The term “resorption” or “bone resorption” refers to the normal processof bone erosion by a group of cells known as osteoclasts. In remodeling,the bone building cells, osteoblasts, infiltrate and fill the resorptionsites to form functional bone. Normally, bone resorption equals boneformation. However, in certain diseases and disorders bone resorptionsurpasses bone formation and bone loss occurs, increasing an individualsrisk for osteoporosis and related bone-weakening diseases.

The term “anti-resorptive agent” as used herein refers to a compound, ordrug having the ability to prevent, delay or reduce resorption of boneor of an implant. Examples of anti-resorptive agents includephosphonates, preferably bisphosphonates and derivatives thereof. Otheranti-resorptive agents include estrogens, prostaglandins and calcitonin.

The terms “bisphosphonate” or “bisphosphonic acid” as used herein,relate to those phosphonates or phosphonic acids that have twophosphonate groups attached to the same carbon atom. Based upon theirchemical composition bisphosphonates can be classified into nitrogencontaining, N-bisphosphonates, including alendronate, zolendronate andpamidronate and non-nitrogen containing bisphosphonates includingclodronate and etidronate. Bisphosphonates are potent inhibitors of boneresorption and are effective in treating diseases and disorders of boneresorption. Many bisphosphonates are known in the art, exhibitingdifferent anti-resorptive potency that can be exploited in thepreparation of the composite. For example, risedronate and zolendronate(contain a nitrogen atom in heterocyclic side group) are more potentthan etidronate in inhibiting bone erosion. It is to be understood thatthe bisphosphonates useful herein include as non-limiting examples,e.g., alendronate, clodronate (clodrinate), etidronate, pamidronate,medronate, nedrinate, tiludronate, zolendronate or combinations thereof.Other non-limiting examples of bisphosphonates have been disclosed inU.S. Pat. Nos. 5,856,314 and 5,338,731.

Furthermore certain bisphosphonate compounds have anti-tumor activityper se (reviewed in Cancer Treat Rev 28(6):305-19, 2002). This featurerenders them useful for thetreatment of bone metastases in certaincancers. The term “osteolysis” refers to the dissolution of bone.

According to one embodiment the present invention provides a compositecomprising synthetic apatite and at least one anti-resorptive agent.According to another embodiment the at least one anti-resorptive agentis a bisphosphonate or bisphosphonate derivative or salt or esterthereof. According to yet another embodiment the present inventionprovides a composite comprising synthetic apatite and at least oneanti-resorptive agent wherein the synthetic apatite is a poorlycrystalline apatite and the at least one anti-resorptive agent is abisphosphonate or bisphosphonate derivative or salt or ester thereof.

In certain circumstances local administration of bisphosphonate ispreferred over systemic delivery. Non-limiting examples includemaintaining integrity of a prosthesis and treating or preventingperiprosthetic bone resorption. Another exemplary indication is toprevent local bone loss due to absence of weight bearing resulting frominjury or trauma. Systemic administration of bisphosphonates to patientshaving bone loss due to the absence of weight bearing resulting from abone injury is well known in the art Patients with renal disease,digestive disorders and other indications may not be candidates forsystemic delivery of bisphosphonates and would benefit from localdelivery.

Therapeutic Agents

According to one embodiment the present invention provides a compositecomprising synthetic apatite and at least one supplementary bioactiveagent selected from a biocompatible polymer and an anti-resorptive agentfurther comprising at least one therapeutic agent including drugs suchas antibiotics and antiviral agents; chemotherapeutic agents;anti-rejection agents; analgesics and analgesic combinations;anti-inflammatory agents; hormones such as steroids and growth factorssuch as fibroblast growth factor. Further provided by the presentinvention is a composition comprising the composite impregnated with adrug or agent able to deliver high tissue levels of said drug to thesite of injured or diseased bone or to a tissue associated with bone. Acomposite of this type is particularly useful for, but not limited to,delivering antibiotic therapy to osteomyelitis patients.

According to another embodiment of the present invention, the at leastone therapeutic agent include cytokines, growth factors and theiractivators etc. for example, in order to enhance a therapeutic effect orto provide a slow-release or sustained-release mechanism. For example,growth factors, structural proteins or cytokines which enhance thetemporal sequence of bone repair, alter the rate of proliferation orincrease the metabolic synthesis of extracellular matrix proteins areuseful additives to the composite of the present invention.Representative proteins include bone growth factors (BMPs, IGF)including BMP2 and BMP7 and fibroblast growth factors, including FGF2,FGF4, FGF9 and FGF18 and variants thereof for bone and cartilagehealing. Other factors shown to act on cells forming bone includeretinoids, growth hormone (GH), leptin and transferrin. Othertherapeutic agents intended to be incorporated in the present inventioninclude blood factors that regulate clot formation such as fibrin andplasminogen.

The proteins of the invention are polypeptides or derivatives, muteinsor variants thereof, obtained from natural, synthetic or recombinantsources, which exhibit the ability to stimulate DNA synthesis and celldivision in vitro of a variety of cells, particularly cell typesinvolved in bone regeneration and remodeling. A non-limiting example ofFGF variants is disclosed in WO 02/36732.

Additionally, cells genetically engineered to express the aforementionedproteins are including in the present invention. Preferred examples forbone repair uses periosteal or other mesenchymal stem cells orosteocytes/osteoblasts per se or transfected with bone growth factorgenes selected from a group including bone morphogenetic protein (BMP)family genes or fibroblast growth factor (FGF) family genes. Accordingto one currently preferred embodiment of the present invention thecomposite comprises at least one growth factor of the FGF family havingosteoinductive activity. According to onecurrently more preferredembodiment of the present invention the composite further comprises agrowth factor of the BMP family.

According to one embodiment the composite comprising synthetic apatiteand at least one anti-resorptive agent further comprise at least onetherapeutic agent selected from antibiotics and antiviral agents;chemotherapeutic agents; anti-rejection agents; analgesics and analgesiccombinations; anti-inflammatory agents or hormones such as steroids.

The mineral component of bone is predominantly made of calciumphosphate. “Hydroxy apatite” refers to a highly crystalline calciumphosphate having the chemical formula: Ca₅(PO₄)₃OH. Hydroxy apatite andother highly crystalline synthetic apatite materials are considered tobe less than optimal bone substitute implants since, although they arebiocompatible, they are for the most poorly biodegradable. The mineralfraction of natural bone is primarily composed of “poorly crystallineapatite”, a calcium phosphate derivative. It is to be understood that aportion of the calcium (Ca⁺⁺) ions may be replaced with other divalentions selected from the group consisting of Magnesium (Mg⁺⁺) and Zinc(Zn⁺⁺). The incorporation of additional or different divalent ionsimparts on the composition certain properties that may be advantageousto bone repair and growth.

Matrix Preparation

According to one embodiment the present invention provides apharmaceutical composition comprising synthetic apatite and at least onesupplementary bioactive agent selected from a biocompatible polymer andan anti-resorptive agent further comprising a pharmaceuticallyacceptable carrier or excipient. According to one embodiment of thepresent invention the pharmaceutical composition further comprises atleast one therapeutic agent.

According to one embodiment of the present invention a pharmaceuticalcomposition comprising synthetic apatite and heparin composite, at leastone carrier having sufficient fluidity to enable injection of thecomposition to the site of treatment and at least one therapeutic agentselected from the group consisting of growth factors and their variantsis provided. According to one currently most preferred embodiment of thepresent invention the at least one therapeutic agent is selected fromthe group consisting of fibroblast growth factors and their variants.

The pharmaceutical composition of the present invention is useful fortreating orthopedic, periodontal and craniofacial indications whereinthere is need to fill a void in a bone including fractures, non-unionfractures, spinal fusion and other indications. According to certainembodiments the pharmaceutical composition is useful for the delivery oftherapeutic agents to a bone lesion or defect. According to otherembodiments, the pharmaceutical composition of the present invention isuseful for cementing prostheses or to prevent osteolysis. Thepharmaceutical composition of this invention may be administered as apaste, preferably as an injectable paste, more preferably as aninjectable paste that hardens in situ, within 24 hours followingimplantation. Alternatively, an implant comprising the composite of theinvention is provided. Furthermore, the composite may be used as acement for or as a coating on synthetic or other implants such as pinsand plates, for example, in hip replacement procedures. Thus, thepresent invention further provides implants or medical devices coatedwith the matrix of the invention.

In accordance with the invention, provided is the use of the compositeof the invention for the manufacture of a medicament for treatingdiseased or injured bone in orthopedic, periodontal and craniofacialindications wherein the composite is provided alone or comprisingtherapeutic agents that accelerate the healing rate and enhance thequality of bone formation.

In certain applications, a solid implant is desired. According to oneembodiment the present invention a bone substitute compositioncomprising synthetic apatite and at least one supplementary bioactiveagent selected from a biocompatible polymer and an anti-resorptiveagent, optionally further comprising a therapeutic agent, optionallyfurther comprising an additive that promotes hardening of saidcomposition in situ over about 10 minutes to about 120 minutes followingapplication. Alternatively, in the reconstruction of structural tissueslike cartilage and bone, certain applications may require implantationof a solid implant. This may be achieved by molding or pressing thecomposition into three dimensional configuration articles of varyingthickness and shape in vitro. Accordingly, provided is an implantcomprising synthetic apatite at least one supplementary bioactive agentselected from a biocompatible polymer and an anti-resorptive agent,optionally further comprising a therapeutic agent, further comprising anadditive that promotes hardening of said composition which may be formedto assume a shape which may constitute a prosthesis. A non-limitingexample of a hardener includes calcium sulfate or calcium phosphatecompounds. The shape is determined by the shape of a mold or supportwhich may be made of any inert material and may be in contact with thecomposite on all sides, as for a sphere or cube, or on a limited numberof sides as for a sheet.

According to one embodiment of the present invention a compositioncomprising said synthetic apatite is prepared for administrationcomprising sterilizing the powder, adding a sufficient amount of liquidto hydrate and disperse the powder, and preparing the wetted powder foradministration. Following the wetting procedure the composition may beoptionally filtered to remove excess liquid, thus leaving a paste-likematerial on the filter.

Further provided is a method of preparing the bone-enhancing composite.The method comprises the following steps:

-   -   a) preparing a liquid mixture comprising ionic calcium,        phosphate, at least one amino acid in either monomeric or        polymeric form, carbonate, at least one supplementary bioactive        agent selected from a biocompatible polymer and an        anti-resorptive agent, optionally further comprising a        therapeutic agent;    -   b) subjecting said mixture to microwave irradiation;    -   c) quenching said irradiated mixture;    -   d) filtering said quenched mixture so as to separate between the        filtrate and a cake;    -   e) drying said cake;    -   f) grinding said cake into a powder.

According to one embodiment of the present invention the above processmay further comprise the following steps:

-   -   g) sterilizing said powder;    -   h) wetting said sterilized powder with a solution optionally        comprising at least one therapeutic agent;    -   i) preparing said wetted powder for administration.

The mixture of step a) comprises a calcium ion that may be, for example,calcium chloride added to a concentration of about 5×10⁻³ to about5×10⁻². The phosphate may be a phosphate such as NaH₂PO, added to aconcentration of about 3×10⁻³ to about 2×10⁻². The preferredconcentration is about 6×10−³. The amino acid may be any monomeric orpolymeric amino acid but is preferably L-aspartic acid, added to aconcentration of about 10 ppm to about 50 ppm. The carbonate may be, forexample NaHCO₃, added to a concentration of about 1 ppm to about 600ppm. According to one embodiment, the concentration of carbonate isabout 150 ppm. The supplementary bioactive agent is selected from abiocompatible polymer and an anti-resorptive agent.

According to one embodiment of the present invention the powderresulting from step (f) consists of poorly crystalline apatite (PCA)calcium phosphate aggregates having approximately 0.45 μm to about 10 μmin diameter, preferably about 1 μm to about 6 μm indiameter.

The microwave heating step is typically carried out in a standardkitchen 700W-1000W microwave for approximately 10 minutes to about 30minutes.

The powder of step (f) following autoclave and drying, was shown to be agraft material for filling holes in bones (Ben-Bassat et al, inBiomaterials Engineering and Devices: Human Applications v2, p155-169,2000). The present invention provides a unique composite with superiorproperties useful as a bone-enhancing agent in indications where a bonegrowth or bone enhancement is needed. It is to be understood that theentire process may be scaled up for mass production.

The composition may be sterilized for use in vivo, in particular for usein clinical and therapeutic applications in mammals. The presentinventors have shown that the dry synthetic apatite composite powder issterilizable by ionization, preferably γ-irradiation, and retains itsoriginal X-ray diffraction pattern. The powder resulting from step (f)was irradiated at a minimum of 2.5 Mrad according to known GMPproduction procedures, followed by X-ray diffraction analysis.

Following thermal sterilization, e.g. 140° C. for about 30 minutes toabout 2 hours, the X-ray diffraction pattern shows two minor reflectionswhich may be seen at approximately 2θ at about 32.1° and about 32.7°.According to one embodiment of the present invention, a thermallysterilized composite is useful as bone substitute.

According to one embodiment the present invention provides wetting thesterilized PCA powder with a pharmaceutically acceptable liquid such aswater or a physiological fluid preferably comprising a growth factorother therapeutic agent. The liquid is added in a sufficient amount toallow wetting and dispersion of the powder to form a wetted mixturehaving the consistency of a paste or putty. In one embodiment of thepresent invention the powder is mixed with liquid, such as PBS orhyaluronic acid solution, in a ratio of about 1:1 w/w or w/v to yield apaste-like substance. In one particular exemplary embodiment 0.3 gmpowder is mixed with 0.3 ml sterile water comprising growth factor, inparticular an FGF or FGF variant to yield approximately 0.5 mlpaste-like composition.

Alternatively, a sufficient amount of liquid is added to permit wettingand dispersion of the powder to form a hydrated precursor mixture havinga consistency compatible with application to a filtration device. Thewetted powder is filtered through a sterile filtration device havingpore size enabling retention of the crystalline aggregates on thefilter. Preferably, the pore size of the filtration device permits fullretention of the bone substitute material. In one particular exemplaryembodiment 0.3 gm synthetic apatite composite are mixed with 2 mlsterile PBS comprising a growth factor, in particular an FGF or FGFvariant. The mixture was left for 1 hour to allow the FGF to adsorb tothe composite and the slurry filtered through a 0.45 μm filter to yieldapproximately 0.5 ml paste-like material.

The filtered material is left sufficiently wetted in order to enablehandling without fragmentation or crumbling or separation of the liquidfrom the solid phase. Preferably the wetted powder has a consistency ofputty or paste. Preferably the paste has a viscosity in the range ofabout 10 poises to about 500 poises, preferably about 30 poises to about200 poises.

In another embodiment of the present invention, the wetted powder isblended under sterile conditions to a consistency compatible withadministration to a lesion. The paste may be administered manually orwith a spreading instrument such as a spatula. More preferably, thewetted powder is inserted into a syringe and is prepared for localadministration or injection into the site of the defect or lesion.

The term “therapeutic” refers to any pharmaceutical, drug orprophylactic agent which may be used in the treatment (including theprevention, diagnosis, alleviation, or cure) of a malady, affliction,disease or injury in a patient.

The term “excipient” as used herein refers to an inert substance addedto a pharmaceutical composition to further facilitate administration ofa compound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, or gelatin. Pharmaceutical compositions may alsoinclude one or more additional active ingredients.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, grinding, pulverizing, dragee-making,levigating, emulsifying, encapsulating, entrapping or lyophilizingprocesses.

The term “physiologically acceptable liquid carrier” or “diluent” refersto an aqueous or non-aqueous fluid that is well suited forpharmaceutical preparations.

The composite of the experiment may be used in particle or powder form,or may be combined with a physiological liquid for use as a paste-likematerial. The composition may further comprise hardening agents for insitu or in vivo hardening. Alternatively an implant comprising thecomposite of the invention is provided. Furthermore, the composite maybe used as a coating on synthetic or other implants such as pins andplates, for example, in hip replacement procedures. Thus, the presentinvention further provides implants or medical devices coated with thematrix of the invention.

According to an alternative embodiment the pharmaceutical compositionfurther comprises at least one agent that renders the compositionnon-fluid under physiological conditions. A non-limiting example of acollagen that gels at physiological temperatures is disclosed in WO00/47130.

Bone Repair

Fractures and other defects in long bones heal via a process known asendochondral ossification while defects and lesions in intramembranousbones heal via an osteogenic route (Rabie, et al., Int J Oral MaxillofacSurg 25(5):383, 1996). Four stages of fracture repair have beencharacterized (reviewed in Bolander, Proc. Soc. Exp. Biol. Med. 200(2):165, 1992). Stage 1 is the immediate injury response; stage 2 marks thesynthesis of new bone matrix and callus formation in a process termedintramembranous ossification; stage 3, designated chondrogenesis, occursas the mesenchymal cells develop into chondrocytes and are eventuallyreplaced by cartilage; stage 4 is the formation of bone from cartilagein a process known as endochondral ossification.

According to the principles of the present invention the composite ofthe invention is useful in indications where bone enhancement and bonehealing is desired. According to one embodiment the composite of theinvention is useful in orthopedic indications including periodontalsurgery, and plastic and craniofacial surgery. In a non-limitingexample, the composite of the present invention is useful foraugmentation of the alveolar ridge to facilitate retention of dentureand to fill various periodontal lesions. It is also useful to fill thegap in cases of bony defects, cysts and traumatic bone loss. Thecomposite of the present invention is useful for maxillofacialdysplasia, filling of bone defects in skull, zygomatic and mandibularareas and augmentation of various bony areas. In addition, the compositeof the present invention is useful to reconstruct the calvaria includingrepair of cranial base and temporal bone defects following surgery.Orthopedic applications in which the compositions of the invention areuseful include, but are not limited to, fractures and non-unionfractures resulting from a trauma or generated by surgical means, spinalfusion, hip resurfacing or bone augmentation in indications such asosteopenia or osteoporosis.

According to the principles of the present invention the compositecomprises therapeutic agents that have the capacity to act at some orall of the stages in order to enhance bone repair and ensure theformation of functional bone.

The present invention further provides use of a composite comprisingsynthetic apatite and at least one supplementary bioactive agentselected from a biocompatible polymer and an anti-resorption agent addedab initio, wherein the synthetic apatite comprises ionic calcium,phosphate, carbonate and at least one amino acid in monomeric orpolymeric form for the manufacture of a bone-enhancing medicament.

Kits

The present invention further provides a kit comprising the composite ofthe present invention, where the dry and liquid components may bepresent in separate containers in the kit, or some of the components maybe combined into one container.

The following examples are intended to be merely illustrative in natureand to be construed in a non-limitative fashion.

EXAMPLES Example 1 Preparation of Bone Raft Powder

A method for preparation of a bone substitute is disclosed in U.S. Pat.No. 6,231,607. The bone substitute prepared by this method has an X-raydiffraction pattern similar to natural bone. The present inventors nowdisclose that at least one supplementary agent such as a biocompatiblepolymer and/or an anti-resorptive agent as may be added during theformation of the synthetic apatite crystals. The powder can be used perse or formulated into a fluid composition having advantageous propertiesfor use as a bone graft material.

Materials and Methods

Graduated bottles, 2-liter capacity and 500 ml capacity

Glass ice bath

Microwave Oven (700W)

Vacuum Filter

Millipore Filter 0.45 μm-9 cm diameter Z29078-5 (Sigma)

Microwave oven

Trizma buffer PRE-SET Crystals Type 7.4-FT (Sigma)

Sodium dihydrogen phosphate monohydrate NaH₂PO₄ . H₂O (MERCK)

Sodium Bicarbonate NaHCO₃ (ICN)

L-Aspartic Acid monosodium salt (Sigma)

Calcium Chloride Dihydrate CaCl₂. 2H₂O (MERCK)

DDW/TDW, Filtered.

Two solutions were prepared:

Solution I (CaCl₂+Trizma+supplementary agent): 20.0 gr. Trizma, 2.94 gr.CaCl₂, polymer, 2.0 liters of DDW.

Solution II (NaH₂PO₄+Trizma): 20.0 gr. Trizma, 1.66 gr NaH₂PO₄.H₂O, 0.6gr. NaHCO₃ 0.1 gr. L-Aspartic Acid, 2.0 liters of DDW.

Equal volumes of solution I and solution II (1.5 liters each) were mixedrapidly in a 4 liter glass bowl. The final solution had the followingconcentrations: 0.01 M CaCl₂, 0.006 M NaH₂PO₄, 150 ppm (mg/Liter)NaHCO₃, 25 ppm L-Aspartic Acid with varying concentration ofbiocompatible polymer or anti-resorptive agent.

The solution was heated in a microwave oven at maximum power for 30 min.

The irradiated mixture was placed in an ice bath for 1 hr.

The mixture was filtered through a Millipore filter (0.45 μm).

The precipitate was washed with 50 ml of DDW.

The precipitate was transferred to a glass beaker and dried overnight atabout 55° C.-60° C.

The dried precipitate was ground to a fine powder using a mortar andpestle.

The resulting powder was weighed for quantity determination.

The powder was sterilized by γ irradiation.

The powder was stored in a closed 15 or 50 ml screw capped vials andlabeled according to date, batch number and quantity.

The supplementary bioactive agents were added to solution 1 in theamounts presented in Table 1. The quantities listed do not represent thefinal concentration but rather the amounts added to the startingsolution. TABLE 1 Supplementary agent ug/ml (microgram/ml) Heparin 15kDa 100 50 5 0.5 Heparin 6 kDa 100 50 5 1 0.5 0.25 0.1 0.05 0.001 0.0001SOS 3.33 0.33 0.033 Dex Sulfate 1 Alendronate, acid 1 0.1 0.001Alendronate, Sodium salt 1 0.1 0.001Heparin -MW 15 kDa; heparin-MW 6 kDa; sucrose octasulfate (SOS) MW-1.16kDa; dextran sulfate MW 10 kDa. The composites are tested in adsorption,resorption and release assays, in X-ray diffraction and FITR. (kD or kDarefers to kilodalton)

Example 2 X-Ray Diffraction Analysis

Bone substitute, material prepared according to U.S. Pat. No. 6,231,607and composite prepared according to Example 1 and sterilized by heatsterilization or γ-irradiation was exposed to X-ray. An X-raydiffraction (XRD) pattern was obtained from a packed powder sample ofthe material pulverized in a mortar and pestle. X-rays were performedusing an X-ray powder diffractometer, Rigaku, Japan. The scan rate wasset to 0.5 degree/minute over the 2 theta (2θ) angular range from20°-35°. The step size was set to 0.05°. FIG. 1A shows the X-raydiffraction pattern of the bone substitute powder prepared according toU.S. Pat. No. 6,231,607. A peak at 2θ about 26° and a largeundifferentiated peak at 2θ about 31° to about 33° are noticeable. TheX-ray diffraction pattern of a composite comprising heparin, wherein theheparin was added to 0.5 parts ug/ml is presented in FIG. 1B. The X-raydiffraction pattern of a composite comprising dextran sulfate added to0.5 ug/ml is shown in FIG. 1C. The X-ray diffraction patterns ofcomposites comprising sucrose octasulfate added to 0.5 or 5 ug/nl areshown in FIGS. 1D and 1E, respectively. In all the cases the X-raydiffraction patterns of the composites show strong similarity to theunradiated material, specifically a characteristic peak at 2 theta ofabout 25.5° to about 26° and an undifferentiated peak at 2 theta 2θ ofabout 31° to about 33°.

The X-ray diffraction pattern of a composite prepared with 0.0005 ugalendronate/ml is presented in FIG. 1F. The acid form and the sodiumsalt form gave identical results. The X-ray diffraction pattern of acomposite prepared with 0.05 alendronate or 0.5 ug alendronate/ml areshown in FIGS. 1G and 1H respectively. The composite comprising thehigher concentration of alendronate (0.5 ug/ml) exhibits a highcrystallinity pattern with an additional peak at 2 theta 2θ=24.24° and alarger peak around 2 theta 2θ=25.9°.

Example 3 Scanning Electron Microscope Analysis

Bone substitute composites prepared with various amounts of differentpolysaccharides was subject to Scanning Electron Microscope Analysis(SEM). FIGS. 2B and 2C show the aggregate formation of the syntheticapatite-heparin composite (0.5 ug/ml and 100 ug/ml, respectively) ascompared to the synthetic apatite (PCA) alone shown in FIG. 2A. Thecomposite prepared from the liquid mixture comprising 0.5% W/W HeparinMW 6,000 exhibited a distinct structure as seen by SEM. All figures are39570× magnification.

Example 4 Bone Substitute Composite with a Poloxamer Copolymer

Pluronic® is a block co-polymer that may serve to impart select physicaland biological properties to the composite, serves as a hemostaticagent. The formation of this system is carried out essentially followingthe steps as for the biocompatible polymers exemplified above.

The synthetic apatite-heparin composite was mixed with varyingconcentrations of Pluronic, 1:500 to about 1:16,000 weight ratios ofPluronic to composite. 100 μ rod shaped structures of the blend,comprising an FGF (5 and 10 μg/ml), were examined for FGF release in aFDCP assay. At concentrations of 1:2000 and less Pluronic had nodetrimental effect on cell proliferation activity of FGF. Thecomposition is further tested in animal models of fracture healing.

Example 5 Resorbability Assay

Assays for determining the rate of in vivo biodegradability includeintramuscular, subcutaneous and intraosseous models. An in vivo assaymeasuring subcutaneous resorption of dense carbonate apatite isdisclosed in Barralet (Barralet, J. et al., J Biomed Mater Res49(2):176-82, 2000). Another example discloses the resorption of porousceramic implants in a dog model (Pollick, S., et al., J Oral MaxillofacSurg, 53(8):915-22; 1995). Other in vivo assays include those that arepresented in Example 10 herein.

Example 6 Semi-Fluid Composition

These examples demonstrate the methods of preparing a semi-fluidcomposition for use as a bone enhancing material.

An amount of 0.3 gm dry sterile powder of Step 1 was mixed with 2 mlsterile PBS. The composition was mixed for 1 hour on a shaker andfiltered through a 0.45 μm membrane to remove excess liquid. Remainingon the filter was approximately 0.5 ml of a pasty substance that wasplaced into a syringe for local administration in an animal model, forexample as is described in example 10, vide infra.

An amount of 3 gm dry sterile powder of Step 1 was mixed with 3 mlsterile PBS to yield approximately 5 ml of a pasty substance that wasplaced into a syringe for local administration in an animal model.

Additional compositions are prepared by varying the w/w or w/v ratio ofthe composite and a pharmaceutically acceptable diluent includinghyaluronic acid. Viscosity of the fluid or semi-fluid compositions wasdetermined by standard techniques.

Example 7 FGFR-Transfected FDCP Proliferation Assay

This assay was used to determine the release of FGF from the compositecomprising the synthetic apatite and a polymer, specifically heparin,SOS or dextran sulfate.

The FDCP cell line is a murine immortalized, interleukin 3(IL-3)-dependent cell line of myelocytic bone marrow origin that doesnot express endogenous FGF Receptors (FGFR). Upon transfection with FGFRcDNA, the FDCP cell line exhibited a dose-dependent proliferativeresponse to FGF that replaces the dependence on IL-3. FGFR transfectedFDCP cells can therefore be used to screen for FGFR signaling. The cellresponse to various ligands is quantitated by a cell proliferation assaywith XTT reagent (Cell Proliferation Kit, Biological Industries Co.).Specifically, FDCP cells stably expressing the FGFRI (FDCP-FGFRI) weregrown in “full medium” (Iscove's Medium containing 2 ml glutamine, 10%FCS, 100 ug/ml penicillin, 100 ug/ml streptomycin) supplemented with 5ug/ml heparin. Cells were split every 3 days and kept in culture no morethan one month. One day before the experiment the cells were split.Before the experiment the cells were washed 3 times (1000 rpm, 6 min)with full medium. The cells were resuspended and counted with TrypanBlue. Twenty thousand (2×10⁴) cells were added to each well of 96-wellplate in 50 μl full medium containing heparin. Condition mediumcontaining FGF or FGF complexed with the various polysaccharides wasadded in an additional volume of 50 μl full medium to bring the finalvolume to 100 μl. The plate was incubated for 48 hours at 37° C. To testcell proliferation, 100 μl of PMS reagent was added to 5 ml of XTTreagent and mixed well (according to manufacture protocol). 50 μl of thelatter solution were aliquoted into each well, and the plates incubatedat 37° C. for at least 2 hours and the color developed was read by aspectro-ELISA reader at A_(490nm).

Example 8 Release of FGF from the Composite

Certain FGF molecules were used as the therapeutic agent and both thesynthetic apatite and synthetic apatite-heparin composite were tested ascarriers.

An FGF (FGF2, 200 μl of a 1, 10 or 20 um/ml solution) was added to 50 μlof dry synthetic apatite and synthetic apatite-heparin composite andallowed to adsorb for 1 hour at room temperature (RT). The syntheticapatites were centrifuged, the supernatant removed, and washed with1×PBS. Two independent assays were performed on this material. An ELISA,as described in example 9, was carried out to establish the total amountof FGF that bound to the synthetic apatite and synthetic apatite-heparincomposite. An FDCP proliferation assay, as described in example 7, wascarried out to determine whether the FGF bound to the material remainedactive.

FIG. 3A shows the amount of FGF that was able to bind to either thesynthetic apatite prepared according to U.S. Pat. No. 6,231,607 (PCA)and the various synthetic apatite-polymer composites as determined bydirect binding ELISA.

FIG. 3B shows the results of the proliferation assay of FGFR1 expressingFDCP cells in the presence of composite comprising FGF and suggests thatthe amount of FGF released and the rate of release from the compositedepended on the type of polymer incorporated into the composite. SOSrefers to the synthetic apatite-polymer composite comprising SOS (0.5ug/ml added ab initio). The dextran adsorbed FGF quite poorly (>5%) andit appears that the FGF was released in a regular manner, albeit insmall doses. A majority of the FGF that bound was released within thefirst day. “hep” refers to the synthetic apatite-heparin compositecomprising heparin (MW 6,000; 0.5 ug/ml). A large proportion of the FGFadded to the composite was adsorbed by the composite (85%) and theamount of FGF released was spread out over the 7 weeks of the assay.This results suggests that the synthetic apatite-heparin compositeprovides a good delivery system for controlled release of FGF. It willbe apparent to a person with skill in the art that differentpolysaccharides, preferably glycosaminoglycans including heparinderivatives, including very low molecular weight heparin, low molecularweight heparin, heparin derivatives and heparin mimetics may be used inplace of the heparin tested herein to control release of specific growthfactors to different tissue types. The synthetic apatite itself showedreduced release of FGF over time, despite the fact that 20% of the FGFadded was adsorbed to the material. FIG. 3C shows the release of an FGFfrom a composite comprising heparin of different molecular weights andconcentrations. Greater amounts of FGF are released from a compositecomprising a low molecular weight heparin (6 kD) at higher concentration(0.5-5 ug/ml) than from high molecular weight heparin (15 kD). Differentcomposites may be beneficial for different applications and indications.FIGS. 3D and 3E show the release profile of FGF (160, 320 or 640 ug/ml)from synthetic apatite-heparin composites, comprising either 0.5 ug/mlheparin (3D) or 5 ug/ml heparin (3E). The rate of release of atherapeutic agent the composite may be optimized for differentapplications and tissue types. For example, endochondral bone formationmay benefit from a different release pattern of FGFs thatintramembranous bone formation. In infected tissue, fast release of ananti-microbial and slow release of a growth factor may enhance healing.

Example 9 Direct Binding ELISA

This assay was performed to quantify the binding of a therapeutic agentto the poorly crystalline apatite or to the synthetic apatite and apolymer composite.

For the ELISA assay, 50 ul of sample was dissolved in 1 ml of 250 mMEDTA for 5-7 hours at RT with shaking and measured as follows:

The wells of the plate were coated with an FGF/sodium bicarbonatesolution. A calibration curve was prepared for FGF concentrations in therange of 250 ng/ml to 8 ng/ml. Dilutions of test-samples (PCA orPCA-derivative adsorbed with a therapeutic agent) were prepared inbicarbonate-buffer to get a final concentration of 0.1 M bicarbonate.The dilutions of the test samples were in a range so that the predictedconcentration of FGF fell in the linear area of the calibration.

To each well of a 96 well plate (NUNC immunoplate, F96 maxisorp) 100 μlof each test sample was added. The plate was covered with Parafilm™ andincubated at 4° C. overnight (ON). The wells were washed with 2M NaClonce and with PBS twice. Detection was carried out as follows: the wellswere blocked with 2% BSA by adding 300 μl 2% BSA to each well. Thesamples were incubated for 1 hour at room temperature (R.), or at 4° C.ON. The wells were washed five times with 300 μl PBST (0.5 ml Tween-20to 500 ml PBS). The antibody, 100 μl of DG2 (anti FGF2 1:5,000) wasadded to each well except control. The samples were incubated 2 hours atRT and washed thrice with PBST. One hundred microliter (100 μl)secondary antibody (HRP-conjugated Goat α mouse 1:10,000) was added toeach well followed by 3 washes with PBST. TMB substrate (100 μl) wasadded to each well and the samples incubated at RT until the desiredcolor developed, after about 10 min. and the reaction stopped by adding50 μl 1 M H₂SO₄ to each well. The plate was read in an ELISAspectrophotometer at A_(450nm).

Example 10 Rat Tibia Model

Objectives: To investigate bone in-growth using the bone enhancingcomposites of the present invention. In some examples the composite wascompared to a commercially available ceramic. SA refers to the syntheticapatite alone, SA-hep refers to the synthetic apatite-heparin composite,SA-SOS refers to the synthetic apatite-SOS composite.

Surgical procedure: Animals were anesthetized according to a standardprotocol, using intramuscular (IM) injection of 85 mg/kg ketamine and 3mg/kg hyaline. A defect was created in the proximal tibial metaphysis,3-4 mm below the collateral ligament insertion, by drilling a hole of 2mm diameter and 2-3 mm in depth or by cutting a wedge of approximately1.5 mm deep and 3 mm wide.

The defect was treated according to the treatments listed in the tablesbelow by locally administering various amounts of the synthetic apatiteor synthetic apatite composites of the invention using a 1 ml syringe orapplying a paste.

Quality evaluation: at the end of the experimental procedure, generally4-8 weeks, rats were sacrificed and the defect area evaluatedhistologically for gross cell morphology, cell abundance and theappearance of extra-cellular material. Standard histological stainingmethods were used (H&E) and the tissue samples were scored by apathologist for evaluation of histological changes during the healingprocess.

The following tables show the experimental setup of exemplary trials.

Experiment 1 outlined in Table 2 shows the setup of an 8 week experimentin rats having a wedge defect introduced into the tibia. The effect ofSA alone was compared to the effect of SA-SOS composite and a SA-SOScomposite (SOS, 0.5 ppm) further comprising an FGF variant (FGF2v).TABLE 2 No. tag LT leg RT leg 1 No tag No treatment No treatment 2 Tailcut SA SA 3 RT ear cut SA + SOS SA + SOS 4 ∇ in ear SA + FGF2v SA +FGF2v 5 ∇ in ear SA + FGF2v SA + FGF2v 6 Lt ear cut SA + SOS + FGF2vSA + SOS + FGF2v 7 Lt ear cut SA + SOS + FGF2v SA + SOS + FGF2vFIG. 4A shows the filling in of the wedge defect with new bonesurrounding the SA particles. FIG. 4B shows the bone repair surroundingthe SA-SOS aggregates. FIG. 4B shows the presence of more SA-SOSparticles remaining in the wedge suggesting that the composite hasbetter cohesive properties than the SA alone. The new bone is developingaround the SA-SOS particles suggesting that a composite of the presentinvention comprising synthetic apatite and sucrose octa sulfate (SOS) isa good quality bone enhancement material.

3) Experiment 2 outlined in Table 3, was designed to test the effect ofdifferent concentrations of FGF in a synthetic apatite-heparin compositein a rat tibia wedge model. TABLE 3 No. Tag Lt leg Rt leg 1-2  No tagSA + HEP SA + HEP 3-4  Tail cut SA + HEP + SA + HEP + 0.04 μg FGFV 0.04μg FGFV 5-6  LT ear cut SA + HEP + SA + HEP + 0.2 μg FGFV 0.2 μg FGFV7-8  RT ear cut SA + HEP + SA + HEP + 1 μg FGFV 1 μg FGFV 9-10 2 earsSA + HEP + SA + HEP + 0.2 μg FGFV 0.2 μg FGFV 11-12  No tag SA + HEPSA + HEP 13-14  Tail cut SA + HEP + SA + HEP + 0.04 μg FGFV 0.04 μgFGF2V 15-16  Lt ear cut SA + HEP + SA + HEP + 0.2 μg FGFV 0.2 μg FGFV17-18  Rt ear cut SA + HEP + SA + HEP + 1 μg FGFV 1 μg FGFV

FIGS. 5A and 5B show new bone (NB) formation around the SA-heparinparticles (H) four weeks post op. The arrows outline the site of thecut. The entire wedge is filled withnew bone. New bone was observed inall the treatments. SA-hep particles are still observed four weeks postop.

4) Table 4 shows the experimental setup designed to test anothercomposition comprising the synthetic apatite-heparin composite. Theaddition of gelatin to the composite changed its consistency. Thecomposition was injected into holes made in the rat tibiae. TABLE 4 No.Tag Left leg Right leg 1-2 No tag SA − HEP + Gelatin SA − HEP + Gelatin3-4 Tail cut SA − HEP + SA − HEP + Gelatin + FGFV Gelatin + FGFV 5-6 Rtear cut SA − HEP + SA − HEP + Gelatin + FGFV Gelatin + FGFV 7-8 Lt earcut Com CaPO₄ Com CaPO₄  9-11 both ears Com CaPO₄ + Com CaPO₄ +Gelatin + FGFV Gelatin + FGFV 12-13 No tag SA − HEP + Gelatin SA − HEP +Gelatin 14-15 Tail cut SA − HEP + SA − HEP + Gelatin + FGFV Gelatin +FGFV 16-17 Rt ear cut SA − HEP + SA − HEP + Gelatin + FGFV Gelatin +FGFVCom CaPO₄ = commercially available ceramic calcium phosphate bonesubstitute

FIGS. 6A-6D show the healing effect after 4 weeks, using 3 differentcompositions in the rat tibia model. The arrows demark the extent of thehole. All figures are shown at 40× magnification. NB refers to new bone.FIG. 6A shows new bone formation in an untreated hole, FIG. 6B is newbone surrounding a commercially available calcium phosphate ceramic.FIG. 6C shows the new bone surrounding the SA particles (P) while FIG.6D shows new bone surrounding SA-hep (P-H) particles. FIG. 6D shows fullintegration between new bone and SA-hep particles of the presentinvention.

Example 4 shown in Table 5 was designed to test the effect of a calciumsulfate hardener on administration of the SA composite and its effect onbone repair. TABLE 5 No. Left leg Right leg 1-2 No treatment Notreatment 3-5 SA − HEP + CaSO₄ SA − HEP + CaSO₄ 6-7 SA − HEP + CaSO₄ +SA − HEP + CaSO₄ + 1 μg FGF 1 μg FGF 8-9 SA − HEP + CaSO₄ + SA − HEP +CaSO₄ + 10 μg FGF 10 μg FGF

Calcium sulfate was added to the composition to provide in situhardening of the composition. The calcium sulfate had no detrimentaleffect on bone formation and all the induced holes filled with new bonewithin seven weeks. FIG. 7 shows the healing of the filled defects. FIG.7A shows new bone from a defect filled with an SA-hep calcium sulfatecomposition. FIG. 7B shows new bone from a defect filled with an SA-hepcalcium sulfate composition further comprising FGF.

The experiment was repeated using recombinant BMP2 in place of the FGF,with similar healing results.

Example 11 Rat Calvarial Model

Two rat calvarial defect models are used to determine the efficacy ofthe composition of the invention to induce bone repair of large defects.In one model, two 3 mm defects per calvaria are drilled using a trephineon both sides of the median suture; one side serves as a control. Theprotocol and evaluation method is described in Colombier (Colombier etal ., Cells Tissues Organs 164:131-140, 1999).

The second model, described in Hollinger (Hollinger and Kleinschmidt, JCraniofacial Surg 1:60-68, 1990) introduces an 8 mm defect in theparietal bone. The defect is filled with the composition of theinvention and the incision site sutured. Following 4 weeks the animalsare euthanized and the defect sites recovered. Histological analysisproceeds as in example 12.

Example 12 Non-Union Model

Distraction osteogenesis is a useful method for bone elongation ofextremities in short stature, for example in individuals diagnosed withdifferent forms of dwarfism such as achondroplasia. This process of bonelengthening is long and often complications arise such as non-union orpoor quality of the regenerated bone.

The maximal rate of elongation used in the current procedure of limbelongation, while maintaining proper bone healing and reconstitution isapproximately 1 mm/day. Faster elongation rates have resulted in lack offusion or in the formation of weak bone that breaks easily or is notweight bearing. For this reason, and in order to enable a shorterelongation period with the concomitant formation of strong, healthy boneit is necessary to provide graft substitute that will promote boneregeneration. For optimal comparison to humans, it is important toperform the procedure in a large animal model like sheep, preferablywith analogous devices and elongation techniques. The model is adjustedto test enhancing bone formation at an elongation rate of 1.5 mm/day.

Materials and Methods

Treatment arms:

Treatment 1: 6 lambs (6 limbs), Control no treatment

Treatment 2: 6 lambs (6 limbs), fluid bone substitute of the invention

Treatment 3: 6 lambs (6 limbs): Heparin modified synthetic apatite

Treatment 4: 6 lambs (6 limbs): Heparin modified synthetic apatite withFGF2 variant

Lambs are assigned randomly into one of the two treatment arms. Surgicallengthening of the right femur is performed in 24 sheep aged from 3 to 4months.

Anesthesia and pre-mediation: General anesthesia is given withoutendotracheal intubation. Intramuscular atropine is given aspremedication (0.5 mg/kg), and thiopentone sodium-2.5% (10-15 mg/kg),Fentanyl® (0.0015 mg/kg) and Diazepam® (0.2 mg/kg) is administeredintravenously.

Fixation: A monolateral external fixator (Monotube-Triax®, StrykerTrauma, Geneva, Switzerland) with four pins, two proximal and two distalin each of its pin clamps, are positioned so that the pins are kept awayfrom the growth plates and the surface of the joint. The osteotomy isperformed using a pneumatic saw.

Lengthening: The lengthening procedure begins seven days after surgeryfor all treatment groups and continues until the limb is lengthened by 5cm. The total elongation period lasts 33 days, at a rate of 1.5 mm/day.

Treatment 1

Control—To assess the effect during the consolidation period, animalsundergo surgery but receive no treatment.

Treatment 2

To assess the effect of the bone substitute, the bone substitute isadministered once, one week after completion of elongation.

Treatment 3

To assess the effect of the carrier alone during consolidation periodheparin modified synthetic apatite will be administered once, one weekafter completion of elongation.

Treatment 4

To assess the effect of the product during consolidation period, heparinmodified synthetic apatite with FGF2 variant will be administered once,one week after completion of elongation.

Animals are kept in a limited area, during the extent of the wholeexperiment and will be allowed to feed and walking ad libitum in cage.Animals are weighed at fixed intervals and general well-being ismonitored.

To study bone formation in the host bone, four different bone markersfluorochromes are administered IM, according to the following schedule:one week after surgery: calcein (green, Sigma®); two weeks aftersurgery: alizarin (red, Sigma®); three weeks after surgery: xylenol(orange, Fluka®) and three days before sacrifice oxytetracycline(yellow, Duphacycline®).

Assessment of efficacy: Success is determined by healing and bonequality obtained after elongation.

X-ray: Progress of bone healing will be controlled by X-ray at weeks 1,2 and 4 after beginning of elongation. The parameters to be assessedfrom the X-ray are: degree of callus formation, gap closure andremodeling achieved during treatment. X-ray scoring is performed by anorthopedic surgeon, according to established bone healing gradingsystems.

The Spalteholz technique to analyze the vascularization of thelengthened callus in each group is performed after intraarterialinjection of Berlin blue through the femoral artery before sacrifice.

Completion: The animals are sacrificed three months after initialsurgery by IV injection of 5 meq of KCl, after anesthesia with sodiumpentobarbital (1.5 mg/kg weight).

Histology: The callus is divided into two parts, one for embedding inparaffin, and the other undecalcified for embedding inmethylmethacrylate. For the histological study, the specimens are fixedin Bouin for 24 hours and decalcified in a solution of PVP-EDTA, at 4°C. Once specimens are decalcified, they are dehydrated using alcohols ofincreasing proof (70%, 80%, 96% and 100%), and after 4 hours in xylene,they are embedded in paraffin at a temperature of 60° C. The specimensare sectioned at 4 μm, and stained with Masson's trichrome, hematoxylinand eosin (H&E), safranin-O and von Kossa.

To analyze the mineralization by fluorochromes, the specimens are fixedin formalin for one week, then dehydrated using alcohols of increasingproof. After one week in PMMA-alcohol and three weeks in PMMA (Technovit7200 VLC®), specimens are sectioned with a diamond saw (Exakt®) andtrimmed to a thickness of 14 μm. After measuring the sections withultraviolet light the distance of the bone markers is measured and thebone index formation calculated (distance mm/days)

The proximal tibiae are extracted and cut in lateral and medial parts.The lateral portion is placed in 4% buffered formaldehyde. Afterdecalcification in EDTA, the specimens are embedded in paraffin and cutinto 4 μm slices. The H&E, Masson's trichrome, Safranin-0 and Alcianblue-PAS stains are applied according to standard technique.

Immunohistochemistry: Antibodies to collagen I, collagen II, FGF1, andS-100 are used to detect protein expression in the lengthened callus byan indirect two-step method: 4 μm paraffin sections are trypsinized anddeparaffinized. Endogenous peroxidase is blocked by placing the sectionsin hydrogen peroxidase solution for 30 min. They are incubated in thefollowing reagents with appropriate Tris-buffered-saline (TBS: 0.55 M,pH 7.36) washes: normal pig serum for 30 min, primary antibody for 1hour, secondary biotinylated antibody for 30 min, and avidin-biotincomplex (Dako KO355) for 30 min. The reactions are visualized withchromogen substrate solution (diaminobenzidine, hydrogen peroxidase, TB)and sections are counterstained with Harris's hematoxylin, dehydrated,and mounted. As anegative control, TBS is used instead of the primaryantibodies. All stained sections are examined and photographed with useof a microscope (Nikon Optiphot-2®, Japan).

Morphometric analysis: An image analyzing system (Leica Q 500MC®)determines the histomorphometric parameters. The parameters determinedusing Masson's trichrome stain are: Trabecular width; Trabecular area;Trabecular erosion surface; Index of trabecular erosion; Number ofosteoblasts; Number of osteoclasts per field; Number of osteoclastnuclei; Index of bone resorption or number osteoclastnuclei/osteoclasts.

The parameters determined using von Kossa's stain are: Osteoid width;Osteoid-trabecular index; and the fluorescence staining measures theextent of long bone formation.

Example 13 In Vitro and In Vivo Assays for Synthetic ApatiteBisphosphonate Composite

The synthetic apatite-bisphosphonate composite of the present inventionwas tested for bisphosphonate activity in an in vivo rat model byinjecting either SA or a SA-bisphosphonate semi-fluid compositiondirectly into a hole created in the calvarial bone and filling with thecomposition. The SA-bisphosphonate composite prepared with 1 mg/mlalendronate. The composition was made by mixing equal volumes of thecomposite with PBS. The animals were sacrificed after 7 days.

Histological sections were made and stained for TRAP (Tartrate ResistantAcidic Phosphate), a marker for osteoclasts. TRAP staining is reddish incolor. FIG. 8A shows the sections in which SA alone was injected, FIG.8B shows a section where SA-alendronate was injected. FIG. 8A showsstrong TRAP staining on or near many of the SA particles, indicatedactive bone remodeling around the particles. FIG. 8B shows a sectionwherein SA-alendronate composition was injected into the calvarial hole.No reddish staining was seen in the vicinity of the particles or withinthe area of injury strongly suggesting that the anti-resorptive activityof the bisphosphonate is not hindered in the co-precipitation process.

Additional cell assays and animal models include in vitro osteoclastresorption (i.e. Taylor et al., Int J Oral Maxillofac Implants,17:321-30, 2002), maintenance of alveolar bone following toothextraction (Denissen, et al, J Periodontol, 71:279-86, 2000), repair ofa segmental defect in a canine femur (Fujibayashi et al., J Long TermEff Med Implants; 11:93-103, 2001), inhibition of debris inducedosteolysis caused by prosthesis loosening (Schwarz et al., J Orthop Res,18:849-55, 2000), a mouse model of inflammatory bone remodeling (JARO2:65-71, 2001).

Although the present invention has been described with respect tovarious specific embodiments thereof in order to illustrate it, suchspecifically disclosed embodiments should not be considered limiting.Many other specific embodiments will occur to those skilled in the artbased upon applicants' disclosure herein, and applicants propose to bebound only by the spirit and scope of their invention as defined in theappended claims.

1-71. (canceled)
 72. A bone-enhancing composite comprising syntheticapatite and at least one supplementary bioactive agent selected from abiocompatible polymer and an anti-resorptive agent added ab initio,wherein the synthetic apatite comprises ionic calcium, phosphate,carbonate and at least one amino acid in monomeric or polymeric form.73. The bone-enhancing composite according to claim 72 wherein thebiocompatible polymer is selected from a natural biocompatible polymerand a synthetic biocompatible polymer.
 74. The bone-enhancing compositeaccording to claim 73 wherein said natural polymer is a polysaccharide.75. The bone-enhancing composite according to claim 74 wherein saidpolysaccharide is a glycosaminoglycan.
 76. The bone-enhancing compositeaccording to claim 75 wherein said glycosaminoglycan is heparin or aheparin derivative.
 77. The bone-enhancing composite according to claim72 further comprising at least one therapeutic agent.
 78. Thebone-enhancing composite according to claim 77 wherein the at least onetherapeutic agent is selected from the group consisting of antibiotics,antiviral agents, chemotherapeutic agents, anti-rejection agents,analgesics and analgesic combinations, anti-inflammatory agents,hormones, growth factors and cytokines.
 79. The bone-enhancing compositeaccording to claim 78 wherein said at least one therapeutic agent is agrowth factor.
 80. The bone-enhancing composite according to claim 79wherein said growth factor is a fibroblast growth factor or an activefragment or variant thereof.
 81. The bone-enhancing composite accordingto claim 72 wherein said synthetic apatite is a poorly crystallineapatite.
 82. The bone-enhancing composite according to claim 72 whereinsaid synthetic apatite is a poorly crystalline apatite and said at leastone supplementary bioactive agent is heparin or a heparin derivative.83. The bone-enhancing composite according to claim 82 furthercomprising fibroblast growth factor or an active fragment or variantthereof.
 84. The bone-enhancing composite according to claim 72 whereinthe anti-resorptive agent is a bisphosphonate or a pharmaceuticallyacceptable salt or ester thereof.
 85. The bone-enhancing compositeaccording to claim 81 wherein said poorly crystalline apatite having anX-ray diffraction pattern comprising a peak at a 2 theta value of about26° and an undifferentiated peak at 2 theta values of about 31° to about33°.
 86. A method for treating orthopedic, periodontal and craniofacialindications comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising syntheticapatite and at least one supplementary bioactive agent selected from abiocompatible polymer and an anti-resorptive agent added ab initio,wherein the synthetic apatite comprises ionic calcium, phosphate,carbonate and at least one amino acid in monomeric or polymeric form.87. The method according to claim 86 wherein said biocompatible polymeris a glycosaminoglycan.
 88. The method according to claim 87 whereinsaid glycosaminoglycan is heparin or a heparin derivative.
 89. Themethod according to claim 86 further comprising at least one therapeuticagent.
 90. The method according to claim 89 wherein the at least onetherapeutic agent is selected from the group consisting of antibiotics,antiviral agents, chemotherapeutic agents, anti-rejection agents,analgesics and analgesic combinations, anti-inflammatory agents,hormones, growth factors and cytokines.
 91. The method according toclaim 90 wherein said at least one therapeutic agent is a growth factor.92. The method according to claim 91 wherein said growth factor is afibroblast growth factor or an active fragment or variant thereof. 93.The method according to claim 86 wherein said synthetic apatite is apoorly crystalline apatite and said at least one supplementary bioactiveagent is heparin or a heparin derivative.
 94. The method according toclaim 93 further comprising fibroblast growth factor or an activefragment or variant thereof.
 95. The method according to claim 86wherein the anti-resorptive agent is a bisphosphonate or apharmaceutically acceptable salt or ester thereof.
 96. A method ofpreparing a bone enhancing composite comprising synthetic apatite and atleast one supplementary bioactive agent selected from a biocompatiblepolymer and an anti-resorptive agent added ab initio, wherein thesynthetic apatite comprises ionic calcium, phosphate, carbonate and atleast one amino acid in monomeric or polymeric form, the methodcomprising the steps of: a) preparing a liquid mixture comprising ioniccalcium, phosphate, at least one amino acid in either monomeric orpolymeric form, carbonate, at least one supplementary bioactive agentselected from a biocompatible polymer and an anti-resorptive agent,optionally further comprising a therapeutic agent; b) subjecting saidmixture to microwave irradiation; c) quenching said irradiated mixture;d) filtering said quenched mixture so as to separate between thefiltrate and a cake; e) drying said cake; f) grinding said cake into apowder.
 97. The method according to claim 98 further comprising thefollowing steps: g) sterilizing said powder; h) wetting said sterilizedpowder with a solution optionally comprising at least one therapeuticagent; i) preparing said wetted powder for administration.
 98. Themethod according to claim 96 wherein the biocompatible polymer isheparin or a heparin derivative.
 99. The method according to claim 96further comprising at least one therapeutic agent.
 100. The methodaccording to claim 99 wherein the at least one therapeutic agent isselected from the group consisting of antibiotics, antiviral agents,chemotherapeutic agents, anti-rejection agents, analgesics and analgesiccombinations, anti-inflammatory agents, hormones, growth factors andcytokines.
 101. The method according to claim 100 wherein said at leastone therapeutic agent is a growth factor.
 102. The method according toclaim 101 wherein said growth factor is a fibroblast growth factor or anactive fragment or variant thereof.
 103. The method according to claim96 wherein the anti-resorptive agent is a bisphosphonate or apharmaceutically acceptable salt or ester thereof.
 104. The methodaccording to claim 96 wherein said synthetic apatite is a poorlycrystalline apatite having an X-ray diffraction pattern comprising apeak at a 2 theta value of about 26° and an undifferentiated peak at 2theta values of about 31° to about 33°.